P802: PATTERNS OF T-CELL AUTOREACTIVITY DIFFER BETWEEN PEDIATRIC APLASTIC ANEMIA AND HEPATITIS-ASSOCIATED BONE MARROW FAILURE
Authors: M. Nováková, M. Svatoň, A. Skotnicová, M. Suková, L. Řezníčková, T. Valová, D. Pospíšilová, O. Fabri, P. Švec, J. Trka, O. Hrušák, J. Starý, E. Mejstříková, E. Froňková
Journal: HemaSphere (2022)

Background: Both idiopathic aplastic anemia (AA) and hepatitis-associated bone marrow failure (HABMF) are considered to be autoreactive T cell mediated diseases. This is based on indirect evidence, most often the success of immunosuppression or oligoclonality with T cell repertoire restriction. Aims: Our questions were: Does the composition of T cells and T-cell receptor beta (TRB) repertoire differ between HABMF, BMF without preceding hepatitis (nonHABMF) and normal bone marrow (BM)? Are there any shared clonotypes suggesting common antigenic stimulus? How does immunosuppressive therapy affect T-cells and does this correlate with outcome? Methods: In total 22 pediatric patients diagnosed in 2004-2021 with HABMF were included in the study. Sixteen HABMF patients were treated with immunosuppressive therapy (IST) as a frontline treatment, of which 8 patients did not respond. As a control group, we included 10 nonHABMF patients with AA (n=6) and refractory cytopenia of childhood (n=4), all treated with IST with poor response in 6 patients, as well as 10 samples of healthy donor BM grafts and 8 newborn peripheral blood (NBPB) samples. We performed flow cytometry immunophenotyping at diagnosis (D0) and on day 120 (D120) after initiation of IST in HABMF and nonHABMF patients. We performed sequencing of the TRB gene rearrangements according to the EuroClonality-NGS working group protocols using DNA isolated from these samples and normalized DNA input for TRB library preparation to the equivalent of 20 000 CD3+ cells per sample based on flow cytometry data, if possible. Whole exome sequencing (WES) was performed from diagnostic samples of HABMF patients. Results: Patients with HABMF had a significantly lower proportion of CD3+ T cells in their BM compared to the nonHABMF with the predominance of CD8+ T cells and their activation by expression of HLA-DR observed with flow cytometry. The analysis of TRB repertoire was performed in 22 D0 and 10 corresponding D120 samples of HABMF patients that had undergone IST and we compared the diversity and clonotype composition with the nonHABMF patients as well as the BM graft samples and NBPB. Expansion of individual TRB clonotypes (>5% of all reads) at D0 was observed more frequently in HABMF patients (9 out of 22) compared to the nonHABMF patients (1 out of 10). None of these expanded clonotypes were shared among more patients based on their nucleotide or amino acid sequence. There was no correlation of expanded T cell clone dynamics and response to IST. The diversity of TRB repertoire was reduced in the nonHABMF group after IST, however we did not observe any correlation between the clinical response to the IST and initial TRB diversity in either group of patients. WES did not reveal any pathogenic variants in genes associated with immune dysregulation. Summary/Conclusion: Both the proportion of T cells and T-cell composition in the BM differed between HABMF and nonHABMF patients at diagnosis. Although we observed an expected reduction in the CD3+ cells after IST both in the HABMF and nonHABMF group, there was no significant difference in the TRB repertoire diversity in the HABMF patients between D0 and D120. We did not observe any correlation between the repertoire diversity and clonotype evolution and the clinical response to IST. Our data further support the hypothesis that T-cells play a significant role in both AA and HABMF, but suggest that the pathogenetic mechanism of both entities is different. Supported by NV20-03-00284, NV19-05-00332, UNCE/MED/015, NU20J-07-00028 and GAUK 534120.